Inflammatory diseases affect more than fifty million Americans. As a result of basic research in molecular and cellular immunology over the last ten to fifteen years, approaches to diagnosing, treating and preventing these immunologically-based diseases has been dramatically altered. One example of this is the discovery of an inducible form of the cyclooxygenase enzyme. Constitutive cyclooxygenase (COX), first purified in 1976 and cloned in 1988, functions in the synthesis of prostaglandins (PGs) from arachidonic acid (AA). Three years after its purification, an inducible enzyme with COX activity was identified and given the name COX-2, while constitutive COX was termed COX-1.
COX-2 gene expression is under the control of pro-inflammatory cytokines and growth factors. Thus, the inference is that COX-2 functions in both inflammation and control of cell growth. While COX-2 is inducible in many tissues, it is present constitutively in the brain and spinal cord, where it may function in nerve transmission for pain and fever. The two isoforms of COX are nearly identical in structure but have important differences in substrate and inhibitor selectivity and in their intracellular locations. Protective PGs, which preserve the integrity of the stomach lining and maintain normal renal function in a compromised kidney, are synthesized by COX-1. On the other hand, PGs synthesized by COX-2 in immune cells are central to the inflammatory process.
The discovery of COX-2 has made possible the design of drugs that reduce inflammation without removing the protective PGs in the stomach and kidney made by COX-1. These selective COX-2 inhibitors may not only be anti-inflammatory, but may also be actively beneficial in the prevention and treatment of colon cancer and Alzheimer's disease.
An ideal formulation for the treatment of inflammation would inhibit the induction and activity of COX-2 without affecting the activity of COX-1. Historically, the non-steroidal and steroidal anti-inflammatory drugs used for treatment of inflammation lack the specificity of inhibiting COX-2 without affecting COX-1. Therefore, most anti-inflammatory drugs damage the gastrointestinal system when used for extended periods. Thus, new treatments for inflammation and inflammation-based diseases are urgently needed.
The natural pharmacopoeia of plants and herbs used in traditional medicines for the treatment of inflammatory conditions was recently found to contain COX-2 inhibitors. One such plant is Triptergium wilfordi (TW). This herb, known as Lei Gong Teng in China, has been used to treat patients suffering with rheumatoid arthritis with a 92% efficacy rate. Lei Gong Teng is available in the U.S. and is advertised to support the healthy functioning of bone joints (www.China-Med.net).
Over 60 compounds have been isolated from TW, and many have been identified as having anti-inflammatory and immunosuppressive activity. Representative compounds that have been isolated from TW include triptolide, 16-hydroxytriptolide, triptophenolide, tripdiolide, and celastrol. However, the administration and therapeutic effectiveness of these compounds have generally been limited by their low margins of safety.
Triptolide is one of the active, nonalkaloid principles isolated from TW and possesses an extensive suppressive effect on immune function, especially on T and B lymphocytes. Structurally, triptolide is a member of the group of diterpene triepoxide lactones (FIG. 1). The inhibitory effect is direct and believed to occur through the inhibition of interluken-2 (IL-2) production and IL-2R (receptor) expression (Tao, et al. (1995) J. Pharmacol. Exp. Therap. 272:1305; U.S. Pat. No. 5,500,340 to Lipsky et al. Mar. 19, 1996). Clinical trials show that it significantly inhibits the proliferation of peripheral blood mononuclear cells of rheumatic arthritis patients. After receiving this medication, patients usually indicate that stiffness, walking, and hand strength are improved with a decrease in inflammation index. Although not generally life-threatening, adverse effects of triptolide are relatively common in the clinical setting. Approximately 28% of patients taking this compound show some type of side effects, such as gastrointestinal disturbance, nausea and vomiting, hypotension and edema.
Therefore, while triptolide may be useful as an anti-inflammatory agent, it can be toxic even in clinically effective doses. Other researchers have used the triptolide molecule as a starting point for the synthesis of novel analogs expressing similar immune effects, while exhibiting lower toxicity (U.S. Pat. No. 5,962,516 to Qi et al. Oct. 5, 1999). Rather than modifying the triptolide molecule to achieve greater efficacy and lower toxicity, it is the object of this invention to combine triptolide, or a representative diterpene epoxide lactone, with a second molecule to produce a synergistic effect in the target cell. One such synergistic response would be the inhibition of inducible COX-2.
Leaves or infusions of feverfew, Tanacetum parthenium, have long been used as a folk remedy for the relief of fever, arthritis and migraine headaches. Previous reports using feverfew extracts have suggested interference with arachidonate metabolism as the mechanism behind these pharmacological effects. In one study (Sumner et al. (1992) Biochem. Pharmacol. 43:2313-2320), crude chloroform extracts of fresh feverfew leaves produced dose-dependent inhibition of the generation of thromboxane B2 and leukotriene B4 by ionophore- and chemoattractant-stimulated rat peritoneal leukocytes and human polymorphonuclear leukocytes. Other research has suggested inhibition of platelet aggregation and the platelet release reaction by feverfew extracts (Groenewegen et al. (1986) J. Pharm. Pharmacol. 38:709-712). Numerous publications suggest that the biologically active components of feverfew are sesquiterpene lactones, with parthenolide being the most abundant.
In the literature approximately 25, separate biological effects have been reported for parthenolide. The potential pharmacological activities range from the inhibition of isolated bovine prostaglandin synthetase (Pugh and Sambo (1988) J. Pharm. Pharmacol. 40:743-745) to the prevention of ethanol-induced gastric ulcers in the rat (Tournier et al. (1999) J. Pharm. Pharmacol. 51:215-219). Research at the molecular level has described parthenolide inhibition of nuclear factor kappa B (NF-kB) activation in several cell-based systems (Hehner et al. (1999) J. Immunol. 163:5617-5623; Bork et al. (1997) FEBS Letters 402:85-90) and inhibition of inducibile nitric oxide gene expression in cultured rat aortic smooth muscle cells (Wong and Menendez (1999) Biochem. Biophys. Res. Commun. 262:375-380). While these molecular events may account, in part, for some of the biological actions of parthenolide, there exists no consensus on the exact nature of the underlying mechanism for its anti-inflammatory effects.
Clinically effective doses of parthenolide for migraine prevention are on the order of micrograms per kg body weight daily. Human clinical trials have verified the minimum effective dose for migraine prevention, as well as the associated discomfort of nausea and vomiting associated with use of 125 mg of feverfew extract per day. The feverfew extracts used in these trials generally contained between 0.2 to 0.7 percent parthenolide. Therefore, the minimally effective dose of parthenolide would be estimated to be approximately 250 micrograms per day or 4 micrograms parthenolide per kg body weight. Commercial, standardized preparations of feverfew deliver between 600 to 4000 micrograms parthenolide per daily dose. While more than sufficient to effectively control migraine frequency, it is doubtful that these doses of parthenolide would be sufficient to address inflammatory responses.
Research literature on the in vitro anti-inflammatory effects of parthenolide reports inhibitory constants in the micromolar range. Assuming a volume of distribution greater than several hundred mL per kg and a median resonance time less than 12 hours, these parthenolide concentrations could only be achieved and maintained in vivo with dosing mg amounts of parthenolide per kg bodyweight. While such dosing studies have been performed successfully in laboratory animals, no clinical reports describe similar doses of parthenolide in humans. Based upon these estimates, a clinically successful preparation of parthenolide for inflammatory conditions would be required to deliver at least 15 mg parthenolide/kg-day. However, such relatively high doses of parthenolide would be commercially prohibitive due to the cost of production, even for a therapeutic formulation.
Combinations of botanicals containing triptolide along with other herbs have been use in both traditional and commercial medicine. However, the triptolide content of TW is only 0.1%, leaving 99.9% of the ingredients of TW as undefined. Such a large unknown fraction makes it extremely unlikely that triptolide is a significant factor in the pharmacological response of TW in this formulation. Thus, it would be useful to identify a compound that would specifically enhance the anti-inflammatory effect of triptolide so that it could be used at sufficiently low doses or at current clinical doses with no adverse side effects. The optimal formulation of triptolide for preserving the health of joint tissues, for treating arthritis or other inflammatory conditions has not yet been discovered. A formulation combining triptolide and parthenolide to synergistically inhibit COX-2 and support the normalization of joint function has not yet been described or discovered.
While glucosamine is generally accepted as being effective and safe for treating osteoarthritis, medical intervention into the treatment of degenerative joint diseases is generally restricted to the alleviation of its acute symptoms. Medical doctors generally utilize non-steroidal and steroidal anti-inflammatory drugs for treatment of osteoarthritis. These drugs, however, are not well-adapted for long-term therapy because they not only lack the ability to promote and protect cartilage, they can actually lead to degeneration of cartilage or reduction of its synthesis. Moreover, most non-steroidal, anti-inflammatory drugs damage the gastrointestinal system when used for extended periods. Thus, new treatments for arthritis are urgently needed.
The joint-protective properties of glucosamine would make it an attractive therapeutic agent for osteoarthritis except for two drawbacks: (i) the rate of response to glucosamine treatment is slower than for treatment with anti-inflammatory drugs, and (ii) glucosamine may fail to fulfill the expectation of degenerative remission. In studies comparing glucosamine with non-steroidal anti inflammatory agents, for example, a double-blinded study comparing 1500 mg glucosamine sulfate per day with 1200 mg ibuprofen, demonstrated that pain scores decreased faster during the first two weeks in the ibuprofen patients than in the glucosamine-treated patients. However, the reduction in pain scores continued throughout the trial period in patients receiving glucosamine and the difference between the two groups turned significantly in favor of glucosamine by week eight. Lopes Vaz, A., Double-blind clinical evaluation of the relative efficacy of ibuprofen and glucosamine sulphate in the management of osteoarthritis of the knee in outpatients, 8 Curr. Med Res Opin. 145-149 (1982). Thus, glucosamine may relieve the pain and inflammation of arthritis at a slower rate than the available anti-inflammatory drugs.
An ideal formulation for the normalization of cartilage metabolism or treatment of osteoarthritis would provide adequate chondroprotection with potent anti-inflammatory activity. The optimal dietary supplement for osteoarthritis should enhance the general joint rebuilding qualities offered by glucosamine and attenuate the inflammatory response without introducing any harmful side effects. It should be inexpensively manufactured and comply with all governmental regulations.
However, the currently available glucosamine formulations have not been formulated to optimally attack and alleviate the underlying causes of osteoarthritis and rheumatoid arthritis. Moreover, as with many commercially-available herbal and dietary supplements, the available formulations do not have a history of usage, nor controlled clinical testing, which might ensure their safety and efficacy.
It would be useful to provide a composition that would specifically and synergistically enhance the anti-inflammatory effect of triptolide so that these could be used at sufficiently low doses or at current clinical doses with no adverse side effects.